Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Raik Gruenberg
Date created: 2008-08-10 11:00:00
Date modified: 2015-08-31 04:08:35
pSB1AT3X Freiburg Fusion -> BioFusion conversion plasmid
| Types | DnaRegion |
| Roles | plasmid Plasmid |
| Sequences | BBa_J18906_sequence (Version 1) |
Description
pSB1AK3X shares the same backbone as pSB1AK3 but uses modified prefix and suffix sequences (that do not formally adhere to any BioBrick standard):
| ...part... |
|
These prefix / suffix sequences are designed to translate protein parts from the proposed Fusion (aka Freiburg) format to the older BioFusion format from the Silver lab. Construction vectors with this modified Freiburg prefix / suffix could bring a protein part in frame with BioFusion parts and remove the STOP between the AgeI and SpeI site. Restriction / ligation with AgeI + NaeI can (theoretically) transfer Expression parts into this conversion vector which can then be used for normal BioFusion cloning (at the cost of adding a T G before and after the part).
NaeI is an isoschizomer to NgoMIV but generates blunt ends which should allow for a directional transfer.
See BB formats.
pSB1AK3X is a high copy number plasmid carrying ampicillin and tetracyclin resistance. The replication origin is a pUC19-derived pMB1 (copy number of 100-300 per cell). For the version of this plasmid with a CcdB insert, see BBa_P1010.
The given sequence starts with the modified suffix and ends with the modified prefix. The complete vector sequence is therefore obtained from: part + vector.
See also:
Notes
This plasmid was constructed by PCR and InFusion recombination.1) Three NgoMIV restriction sites within the TetR gene of pSB1AT3 were removed using the QuickChange kit (Qiagen)
2) The pSB1AT3 vector backbone was linearized by PCR, introducing the NgoMIV / AgeI restriction sites
3) The P1010 insert was amplified by PCR, introducing the NgoMIV / AgeI restriction sites
4) The two overlapping PCR products were recombined using the clonetech InFusion kit.
All PCR reactions (except the QickChange one) were performed with a proof-reading polymerase featuring a very low mutation rate. Nevertheless, the vector backbone may contain mutations with respect to pSB1AT3. Only insert and flanks have been verified by sequencing.
Source
constructed from pSB1AT3| Sequence Annotation | Location | Component / Role(s) |
| "crossFusion" suffix "crossFusion" prefix | 1,26 3430,3456 | feature/misc sequence_feature feature/misc sequence_feature |